scholarly journals Mycobacterial HflX is a ribosome splitting factor that mediates antibiotic resistance

2019 ◽  
Vol 117 (1) ◽  
pp. 629-634 ◽  
Author(s):  
Paulami Rudra ◽  
Kelley R. Hurst-Hess ◽  
Katherine L. Cotten ◽  
Andrea Partida-Miranda ◽  
Pallavi Ghosh
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Smegmatis ◽  
Mycobacterium Abscessus ◽  
Macrolide Resistance ◽  
Gtp Hydrolysis ◽  
Ribosomal Subunits ◽  
Mutant Proteins ◽  
Splitting Factor

Antibiotic resistance in bacteria is typically conferred by proteins that function as efflux pumps or enzymes that modify either the drug or the antibiotic target. Here we report an unusual mechanism of resistance to macrolide-lincosamide antibiotics mediated by mycobacterial HflX, a conserved ribosome-associated GTPase. We show that deletion of thehflXgene in the pathogenicMycobacterium abscessus, as well as the nonpathogenicMycobacterium smegmatis, results in hypersensitivity to the macrolide-lincosamide class of antibiotics. Importantly, the level of resistance provided byMab_hflXis equivalent to that conferred byerm41, implying thathflXconstitutes a significant resistance determinant inM. abscessus. We demonstrate that mycobacterial HflX associates with the 50S ribosomal subunits in vivo and can dissociate purified 70S ribosomes in vitro, independent of GTP hydrolysis. The absence of HflX in aΔMs_hflXstrain also results in a significant accumulation of 70S ribosomes upon erythromycin exposure. Finally, a deletion of either the N-terminal or the C-terminal domain of HflX abrogates ribosome splitting and concomitantly abolishes the ability of mutant proteins to mediate antibiotic tolerance. Together, our results suggest a mechanism of macrolide-lincosamide resistance in which the mycobacterial HflX dissociates antibiotic-stalled ribosomes and rescues the bound mRNA. Given the widespread presence ofhflXgenes, we anticipate this as a generalized mechanism of macrolide resistance used by several bacteria.

scholarly journals Substrate Binding Analysis of the 23S rRNA Methyltransferase RrmJ

2004 ◽  
Vol 186 (19) ◽  
pp. 6634-6642 ◽  
Author(s):  
Jutta Hager ◽  
Bart L. Staker ◽  
Ursula Jakob
Keyword(s):  
Structural Model ◽  
Ribosomal Subunit ◽  
Site Directed Mutagenesis ◽  
23S Rrna ◽  
Ribosomal Subunits ◽  
Mutant Proteins ◽  
Step Model ◽  
The Individual

ABSTRACT The 23S rRNA methyltransferase RrmJ (FtsJ) is responsible for the 2′-O methylation of the universally conserved U2552 in the A loop of 23S rRNA. This 23S rRNA modification appears to be critical for ribosome stability, because the absence of functional RrmJ causes the cellular accumulation of the individual ribosomal subunits at the expense of the functional 70S ribosomes. To gain insight into the mechanism of substrate recognition for RrmJ, we performed extensive site-directed mutagenesis of the residues conserved in RrmJ and characterized the mutant proteins both in vivo and in vitro. We identified a positively charged, highly conserved ridge in RrmJ that appears to play a significant role in 23S rRNA binding and methylation. We provide a structural model of how the A loop of the 23S rRNA binds to RrmJ. Based on these modeling studies and the structure of the 50S ribosome, we propose a two-step model where the A loop undocks from the tightly packed 50S ribosomal subunit, allowing RrmJ to gain access to the substrate nucleotide U2552, and where U2552 undergoes base flipping, allowing the enzyme to methylate the 2′-O position of the ribose.

scholarly journals Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP–ASAP1 complex

2007 ◽  
Vol 402 (3) ◽  
pp. 439-447 ◽  
Author(s):  
Ruibai Luo ◽  
Bijan Ahvazi ◽  
Diana Amariei ◽  
Deborah Shroder ◽  
Beatriz Burrola ◽  
...  
Keyword(s):  
Binary Complex ◽  
Gtp Hydrolysis ◽  
Gtpase Activating Proteins ◽  
Steady State Kinetics ◽  
Catalytically Active ◽  
Ras Family ◽  
Src Homology ◽  
Significant Conformational Change

Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a kcat of 57±5 s−1 and a Km of 2.2±0.5 μM determined by steady-state kinetics and a kcat of 56±7 s−1 determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4−), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than Km. Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the kcat. Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP–ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

1994 ◽  
Vol 14 (5) ◽  
pp. 3197-3207
Author(s):  
P R Caron ◽  
P Watt ◽  
J C Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

scholarly journals Effective Treatment of Mycobacterium avium subsp. hominissuis and Mycobacterium abscessus Species Infections in Macrophages, Biofilm, and Mice by Using Liposomal Ciprofloxacin

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
James D. Blanchard ◽  
Valerie Elias ◽  
David Cipolla ◽  
Igor Gonda ◽  
Luiz E. Bermudez
Keyword(s):  
Effective Treatment ◽  
Mouse Models ◽  
Mycobacterium Avium ◽  
Nontuberculous Mycobacteria ◽  
Mycobacterium Abscessus ◽  
Content Type ◽  
Number Of Individuals ◽  
Intranasal Instillation

ABSTRACT Nontuberculous mycobacteria (NTM) affect an increasing number of individuals worldwide. Infection with these organisms is more common in patients with chronic lung conditions, and treatment is challenging. Quinolones, such as ciprofloxacin, have been used to treat patients, but the results have not been encouraging. In this report, we evaluate novel formulations of liposome-encapsulated ciprofloxacin (liposomal ciprofloxacin) in vitro and in vivo. Its efficacy against Mycobacterium avium and Mycobacterium abscessus was examined in macrophages, in biofilms, and in vivo using intranasal instillation mouse models. Liposomal ciprofloxacin was significantly more active than free ciprofloxacin against both pathogens in macrophages and biofilms. When evaluated in vivo, treatment with the liposomal ciprofloxacin formulations was associated with significant decreases in the bacterial loads in the lungs of animals infected with M. avium and M. abscessus. In summary, topical delivery of liposomal ciprofloxacin in the lung at concentrations greater than those achieved in the serum can be effective in the treatment of NTM, and further evaluation is warranted.

scholarly journals Clinical extended-spectrum beta-lactamase antibiotic resistance plasmids have diverse transfer rates and can spread in the absence of antibiotic selection

2019 ◽  
Author(s):  
Fabienne Benz ◽  
Jana S. Huisman ◽  
Erik Bakkeren ◽  
Joana A. Herter ◽  
Tanja Stadler ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Conjugative Transfer ◽  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
Extended Spectrum ◽  
Resistance Plasmids ◽  
Serovar Typhimurium ◽  
Spreading Potential

AbstractHorizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global spread of antibiotic resistance. However, the relative contributions of factors that underlie the spread of clinically relevant plasmids are unclear. Here, we quantified conjugative transfer dynamics of Extended Spectrum Beta-Lactamase (ESBL) producing plasmids in the absence of antibiotics. We showed that clinical Escherichia coli strains natively associated with ESBL-plasmids conjugate efficiently with three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain, reaching final transconjugant frequencies of up to 1% within 24 hours in vitro. The variation of final transconjugant frequencies varied among plasmids, donors and recipients and was better explained by variation in conjugative transfer efficiency than by variable clonal expansion. We identified plasmid-specific genetic factors, specifically the presence/absence of transfer genes, that influenced final transconjugant frequencies. Finally, we investigated plasmid spread within the mouse intestine, demonstrating qualitative agreement between plasmid spread in vitro and in vivo. This suggests a potential for the prediction of plasmid spread in the gut of animals and humans, based on in vitro testing. Altogether, this may allow the identification of resistance plasmids with high spreading potential and help to devise appropriate measures to restrict their spread.

scholarly journals RNA polymerase bypass at sites of dihydrouracil: implications for transcriptional mutagenesis.

1995 ◽  
Vol 15 (12) ◽  
pp. 6729-6735 ◽  
Author(s):  
J Liu ◽  
W Zhou ◽  
P W Doetsch
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerases ◽  
Dna Damages ◽  
Mutant Proteins ◽  
Dna Lesion ◽  
Transcriptional Mutagenesis ◽  
Rapid Generation ◽  
Transition Mutation

Dihydrouracil (DHU) is a major base damage product formed from cytosine following exposure of DNA to ionizing radiation under anoxic conditions. To gain insight into the DNA lesion structural requirements for RNA polymerase arrest or bypass at various DNA damages located on the transcribed strand during elongation, DHU was placed onto promoter-containing DNA templates 20 nucleotides downstream from the transcription start site. In vitro, single-round transcription experiments carried out with SP6 and T7 RNA polymerases revealed that following a brief pause at the DHU site, both enzymes efficiently bypass this lesion with subsequent rapid generation of full-length runoff transcripts. Direct sequence analysis of these transcripts indicated that both RNA polymerases insert primarily adenine opposite to the DHU site, resulting in a G-to-A transition mutation in the lesion bypass product. Such bypass and insertion events at DHU sites (or other types of DNA damages), if they occur in vivo, have a number of important implications for both the repair of such lesions and the DNA damage-induced production of mutant proteins at the level of transcription (transcriptional mutagenesis).

scholarly journals Rifabutin Is Bactericidal against Intracellular and Extracellular Forms of Mycobacterium abscessus

2020 ◽  
Vol 64 (11) ◽  
Author(s):  
Matt D. Johansen ◽  
Wassim Daher ◽  
Françoise Roquet-Banères ◽  
Clément Raynaud ◽  
Matthéo Alcaraz ◽  
...  
Keyword(s):  
Opportunistic Pathogen ◽  
Mycobacterium Abscessus ◽  
Zebrafish Model ◽  
Content Type ◽  
Attractive Option ◽  
Conventional Antibiotics ◽  
Susceptibility Profiles ◽  
Combination Regimens

ABSTRACT Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases. As it is intrinsically resistant to most conventional antibiotics, there is an unmet medical need for effective treatments. Repurposing of clinically validated pharmaceuticals represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. In this context, rifabutin (RFB) has been shown to be active against M. abscessus and has raised renewed interest in using rifamycins for the treatment of M. abscessus pulmonary diseases. Here, we compared the in vitro and in vivo activity of RFB against the smooth and rough variants of M. abscessus, differing in their susceptibility profiles to several drugs and physiopathologial characteristics. While the activity of RFB is greater against rough strains than in smooth strains in vitro, suggesting a role of the glycopeptidolipid layer in susceptibility to RFB, both variants were equally susceptible to RFB inside human macrophages. RFB treatment also led to a reduction in the number and size of intracellular and extracellular mycobacterial cords. Furthermore, RFB was highly effective in a zebrafish model of infection and protected the infected larvae from M. abscessus-induced killing. This was corroborated by a significant reduction in the overall bacterial burden, as well as decreased numbers of abscesses and cords, two major pathophysiological traits in infected zebrafish. This study indicates that RFB is active against M. abscessus both in vitro and in vivo, further supporting its potential usefulness as part of combination regimens targeting this difficult-to-treat mycobacterium.

scholarly journals Etamycin as a Novel Mycobacterium abscessus Inhibitor

2020 ◽  
Vol 21 (18) ◽  
pp. 6908
Author(s):  
Bui Thi Bich Hanh ◽  
Tae Ho Kim ◽  
June-Woo Park ◽  
Da-Gyum Lee ◽  
Jae-Sung Kim ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Standard Of Care ◽  
Mycobacterium Abscessus ◽  
Drug Candidate ◽  
Infection Model ◽  
Excellent Activity ◽  
Further Development ◽  
Clinical Drug

The increase in drug-resistant Mycobacterium abscessus, which has become resistant to existing standard-of-care agents, is a major concern, and new antibacterial agents are strongly needed. In this study, we introduced etamycin that showed an excellent activity against M. abscessus. We found that etamycin significantly inhibited the growth of M. abscessus wild-type strain, three subspecies, and clinical isolates in vitro and inhibited the growth of M. abscessus that resides in macrophages without cytotoxicity. Furthermore, the in vivo efficacy of etamycin in the zebrafish (Danio rerio) infection model was greater than that of clarithromycin, which is recommended as the core agent for treating M. abscessus infections. Thus, we concluded that etamycin is a potential anti-M. abscessus candidate for further development as a clinical drug candidate.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

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